Aerotaxis, the behavioral response of Escherichia coli to oxygen, is mediated by the Aer and Tsr receptors. Aer is an internal receptor, anchored in the middle to the inside of the cytoplasmic membrane. An N-terminal PAS domain has a FAD cofactor and senses redox and energy level in the cytoplasm. The carboxyl half of Aer, also cytoplasmic, has a HAMP domain and a signaling domain that is highly conserved in chemotaxis receptors. Elucidating the Aer signaling mechanism will provide the first in-depth study of signal transduction between PAS and HAMP domains: important sensory modules that are widespread in living systems but not widely studied by other investigators. Specific Aim 1 will investigate the structural relationship of the domains of the Aer homodimer. Based on a structural model, the HAMP domain is proposed to be a four-helix bundle that interfaces directly with the PAS domain to form the input/output module of Aer. The structure of the HAMP domain will be determined by disulfide crosslinking in vivo and a NMR solution structure in collaboration with F. W. Dahlquist. Contact surfaces between the PAS and HAMP domains will be determined using genetic analysis, including allele-specific suppression, surface accessibility measurements, and interactional disulfide mapping. The proximal signaling domain is proposed to be a functionally important substructure that converts the rotational signal from the HAMP domain into a displacement of the signaling domain. Clarifying the structural relationships of the Aer domains will guide the design of experiments to determine the signaling pathway. Specific Aim 2 will investigate the aerotaxis signaling mechanism within an Aer homodimer. Reduction of FAD in the Aer PAS domain is proposed to cause a conformational change that induces rotation of a four-helix HAMP bundle, converting the Aer signal output from the kinase-off state to the kinase-on state. Key residues in the FAD-binding cleft will be identified through in silico analyses, site-specific and random mutagenesis, covalent labeling of residues near the isoalloxazine redox center of FAD (using photolabeled-FAD), and FAD-binding measurements. To clarify the sequence of the signaling pathway, successful intragenic complementation studies will be extended to determine whether mutant PAS domains that are locked in the "on" state, signal through the proximal or cognate HAMP-AS-2 helix, or both. We will also use disulfide cross-linking to determine whether the HAMP domain maintains a stable four-helix bundle in both the on and off signaling state. Together these studies should reveal the critical residues of the signaling pathway. Specific Aim 3 will determine the signal(s) sensed by the Aer PAS domain. Although aerotaxis requires the electron transport system, flavin reductase (Fre) reduces Aer-FAD in vitro. A reconstituted aerotaxis system will be used to investigate the role of Fre and test the hypothesis that Aer can sense cytoplasmic redox potential and NADH/NAD ratios, independent of the electron transport system. Elucidating the signaling mechanism in Aer should provide insight into the sensory mechanisms of medically important PAS and HAMP proteins. Investigations of oxygen sensing in E. coli bacteria identified a PAS domain as the sensory module in the receptor. PAS domains are found in 10,000 proteins from bacteria to humans, including medically important proteins. This study seeks to understand how sensory receptors function. The knowledge gained will help scientists understand how more complex sensory systems monitor oxygen concentration and energy levels in cells like human nerve cells that are easily damaged if oxygen is not maintained at adequate levels.